Linda P. Dwoskin

A novel mechanism of action and potential use for lobeline as a treatment for psychostimulant abuse.

Lobeline, an alkaloidal constituent of Lobelia inflata LINN., has a long history of therapeutic usage ranging from emetic and respiratory stimulant to tobacco smoking cessation agent. Although classified as both an agonist and an antagonist at nicotinic receptors, lobeline has no structural resemblance to nicotine, and structure--function relationships do not suggest a common pharmacophore. Lobeline inhibits nicotine-evoked dopamine release and [3H]nicotine binding, thus acting as a potent antagonist at both alpha3beta2(*) and alpha4beta2(*) neuronal nicotinic receptor subtypes. However, lobeline does not release dopamine from its presynaptic terminal, but appears to induce the metabolism of dopamine intraneuronally. Reevaluation of the mechanism by which lobeline alters dopamine function reveals that its primary mechanism is inhibition of dopamine uptake and promotion of dopamine release from the storage vesicles within the presynaptic terminal, via an interaction with the tetrabenazine-binding site on the vesicular monoamine transporter (VMAT2). Thus, lobeline appears to perturb the fundamental mechanisms of dopamine storage and release. Based on its neurochemical mechanism, the ability of lobeline to functionally antagonize the neurochemical and behavioral effects of the psychostimulants amphetamine and methamphetamine was examined. Lobeline was found to inhibit the amphetamine-induced release of dopamine in vitro, and amphetamine-induced hyperactivity, drug discrimination, and self-administration. However, lobeline does not support self-administration in rats, suggesting a lack of addiction liability. Thus, lobeline may reduce the abuse liability of these psychostimulants. The development of lobeline and lobeline analogs with targeted selectivity at VMAT2 represents a novel class of therapeutic agents having good potential as efficacious treatments for methamphetamine abuse.

Environmental enrichment attenuates locomotor sensitization, but not in vitro dopamine release, induced by amphetamine

Rats were raised from weanling until young adulthood in either an enriched condition (EC) or isolated condition (IC). Following this, the locomotor and rewarding effects of amphetamine were determined using the conditioned place preference (CPP) paradigm. EC rats were more sensitive to the acute locomotor stimulant effect and rewarding effect of amphetamine relative to IC rats. In contrast, EC rats were less sensitive than IC rats to the locomotor sensitization effect obtained across repeated amphetamine injections. To determine the effect of environmental enrichment on alteration of brain dopamine (DA) function induced by amphetamine, the effect of amphetamine on electrically evoked release of DA and dihydroxyphenylacetic acid (DOPAC) was determined in vitro using tissue slices from the nucleus accumbens and striatum of EC and IC rats. No differences between EC and IC rats in release of DA or DOPAC were evident, suggesting that the environmentally induced difference in sensitivity to the behavioral effects of amphetamine involves a neural mechanism extrinsic to the mesolimbic and nigrostriatal terminal field regions.

Prefrontal Cortex and Drug Abuse Vulnerability: Translation to Prevention and Treatment Interventions

Vulnerability to drug abuse is related to both reward seeking and impulsivity, two constructs thought to have a biological basis in the prefrontal cortex (PFC). This review addresses similarities and differences in neuroanatomy, neurochemistry and behavior associated with PFC function in rodents and humans. Emphasis is placed on monoamine and amino acid neurotransmitter systems located in anatomically distinct subregions: medial prefrontal cortex (mPFC); lateral prefrontal cortex (lPFC); anterior cingulate cortex (ACC); and orbitofrontal cortex (OFC). While there are complex interconnections and overlapping functions among these regions, each is thought to be involved in various functions related to health-related risk behaviors and drug abuse vulnerability. Among the various functions implicated, evidence suggests that mPFC is involved in reward processing, attention and drug reinstatement; lPFC is involved in decision-making, behavioral inhibition and attentional gating; ACC is involved in attention, emotional processing and self-monitoring; and OFC is involved in behavioral inhibition, signaling of expected outcomes and reward/punishment sensitivity. Individual differences (e.g., age and sex) influence functioning of these regions, which, in turn, impacts drug abuse vulnerability. Implications for the development of drug abuse prevention and treatment strategies aimed at engaging PFC inhibitory processes that may reduce risk-related behaviors are discussed, including the design of effective public service announcements, cognitive exercises, physical activity, direct current stimulation, feedback control training and pharmacotherapies. A major challenge in drug abuse prevention and treatment rests with improving intervention strategies aimed at strengthening PFC inhibitory systems among at-risk individuals.

Environmental enrichment decreases cell surface expression of the dopamine transporter in rat medial prefrontal cortex.

Rats raised in an enriched environmental condition (EC) exhibit a decreased (35%) maximal velocity (Vmax) of [3H]dopamine (DA) uptake in medial prefrontal cortex (mPFC) compared with rats raised in an impoverished condition (IC); however, no differences between EC and IC groups in Vmax for [3H]DA uptake were found in nucleus accumbens and striatum. Using biotinylation and immunoblotting techniques, the present study examined whether the brain region‐specific decrease in DA transporter (DAT) function is the result of a reduction in DAT cell surface expression. In mPFC, nucleus accumbens and striatum, total DAT immunoreactivity was not different between EC and IC groups. Whereas no differences in cell surface expression of DAT were found in nucleus accumbens and striatum, DAT immunoreactivity in the biotinylated cell surface fraction of mPFC was decreased (39%) in EC compared with IC rats, consistent with the magnitude of the previously observed decrease in Vmax for [3H]DA uptake in mPFC in EC rats. These results suggest that the decrease in DAT cell surface expression in the mPFC may be responsible for decreased DAT function in the mPFC of EC compared with IC rats, and that there is plasticity in the regulatory mechanisms mediating DAT trafficking and function.

Nornicotine is self-administered intravenously by rats

Abstract  Rationale: Nicotine is a tobacco alkaloid known to be important in the acquisition and maintenance of tobacco smoking. However, other constituents in tobacco may contribute to the dependence liability. Objective: The present report sought to determine whether nornicotine, a tobacco alkaloid and metabolite of nicotine, has a reinforcing effect. Methods: Rats were prepared with a jugular catheter, then were allowed to self-administer intravenously either S(–)-nicotine (0.03 mg/kg/infusion), RS(±)-nornicotine (0.3 mg/kg/infusion) or saline using a two-lever operant procedure. The response requirement for each infusion was incremented gradually from a fixed ratio 1 (FR1) to FR5. When responding stabilized on the FR5, other doses of nicotine (0.01 mg/kg/infusion and 0.06 mg/kg/infusion) and nornicotine (0.075, 0.15, and 0.6 mg/kg/infusion) were tested for their ability to control responding. Results: Similar to nicotine, rats self-administered nornicotine significantly above saline control levels. Within the dose ranges tested, both nicotine and nornicotine yielded relatively flat dose–response functions. Extinction of responding was evident when saline was substituted for nornicotine, and responding was reinstated when nornicotine again was available. The rate of nornicotine self-administration was similar between rats tested with either 24-h or 48-h inter-session intervals. Conclusion: These results indicate that nornicotine contributes to the dependence liability associated with tobacco use.

Contribution of CNS nicotine metabolites to the neuropharmacological effects of nicotine and tobacco smoking

Nicotine, the principal alkaloid in tobacco products, is generally accepted to be the active pharmacological agent responsible for CNS effects resulting from tobacco use. Arguments are presented in this commentary which take issue with this popular dogma, by providing evidence that nicotine metabolites may also be responsible for the CNS effects commonly attributed to nicotine. CNS effects attributed to nicotine include reinforcing effects, mood elevation, arousal, locomotor stimulant effects, and learning and memory enhancement. The reinforcing and locomotor stimulant effects of nicotine have been suggested to be the result of activation of CNS dopaminergic systems, and nicotine-induced modulation of dopaminergic neurotransmission has been studied in detail. Nicotine acts at a family of nicotinic receptor subtypes composed of multiple subunits; however, the exact composition of the subunits in native nicotinic receptors and the functional significance of the receptor subtype diversity are currently unknown. This nicotinic subtype diversity increases the complexity of the potential mechanisms of action of nicotine and its metabolites. Although peripheral metabolism of nicotine has been studied extensively, metabolism in the CNS has not been investigated to any great extent. Recently, studies from our laboratory have demonstrated that several nicotine metabolites are present in the CNS after acute nicotine administration. Moreover, nicotine metabolites are pharmacologically active in neurochemical and behavioral assays. Thus, CNS effects resulting from nicotine exposure may not be due solely to nicotine, but may result, at least in part, from the actions of nicotine metabolites.

Vesicular monoamine transporter 2: Role as a novel target for drug development

In the central nervous, system, vesicular monoamine transporter 2 (VMAT2) is the only transporter that moves cytoplasmic dopamine (DA) into synaptic vesicles for storage and subsequent exocytotic release. Pharmacologically enhancing DA sequenstration by VMAT2, and thus preventing the oxidation of DA in the cytoplasm, may be a strategy for treating diseases such as Parkinsons disease. VMAT2 may also be a novel target for the development of treatments for psychostimulant abuse. This review summarizes the possible role of VMAT2 as a therapeutic target, VMAT2 ligands reported in the literature, and the structure-activity relationship of these ligands, including tetrabenazine analogs, ketanserin analogs, lobeline analogs, and 3-amine-2-phenylpropene analogs. The molecular structure of VMAT2 and its relevance to ligand binding are briefly discussed.

Lobeline Displaces [3H]Dihydrotetrabenazine Binding and Releases [3H]Dopamine from Rat Striatal Synaptic Vesicles: Comparison with d‐Amphetamine

Abstract: Lobeline, an alkaloid from Indian tobacco (Lobelia inflata), is classified as a nicotinic agonist and is currently used as a smoking cessation agent. However, our previous in vitro studies demonstrate that lobeline does not act as a nicotinic agonist but alters presynaptic dopamine (DA) storage by potently inhibiting DA uptake into synaptic vesicles. Recently, d‐amphetamine has been reported to act at the level of the synaptic vesicle to alter presynaptic function. The present in vitro studies further elucidate the mechanism of lobelines action and compare its effects with those of d‐amphetamine. [3H]Dihydrotetrabenazine ([3H]DTBZ), used routinely to probe a high‐affinity binding site on the vesicular monoamine transporter (VMAT2), bound to vesicle membranes from rat striatum with a KD of 1.67 nM and Bmax of 8.68 pmol/mg of protein. Lobeline inhibited [3H]DTBZ binding with an IC50 of 0.90 µM, consistent with its previously reported IC50 of 0.88 µM for inhibition of [3H]DA uptake into vesicles. These results suggest that lobeline specifically interacts with DTBZ sites on VMAT2 to inhibit DA uptake into synaptic vesicles. Interestingly, d‐amphetamine inhibited [3H]DTBZ binding to vesicle membranes with an IC50 of 39.4 µM, a concentration 20 times greater than reported for inhibition of VMAT2 function, suggesting that d‐amphetamine interacts with a different site than lobeline on VMAT2 to inhibit monoamine uptake. Kinetic analysis of [3H]DA release from [3H]DA‐preloaded synaptic vesicles in the absence of drug revealed a t1/2 of 2.12 min. Lobeline and d‐amphetamine evoked [3H]DA release with EC50 values of 25.3 and 2.22 µM, respectively. At a concentration 10 times the EC50, lobeline and d‐amphetamine significantly decreased the t1/2 of [3H]DA release to 1.58 and 1.48 min, respectively. Thus, in contrast to d‐amphetamine, which is equipotent in inhibiting DA uptake and promoting release from the synaptic vesicles, lobeline more potently (28‐fold) inhibits DA uptake (via an interaction with the DTBZ site on VMAT2) than it evokes DA release to redistribute presynaptic DA storage.

Environmental enrichment enhances sensitization to GBR 12935-induced activity and decreases dopamine transporter function in the medial prefrontal cortex

Rats raised in an enriched condition (EC) during development display increased hyperactivity to the effect of acute amphetamine compared to rats raised in an impoverished condition (IC). The present study determined whether environmental enrichment differentially alters the effects of GBR 12935 administration, a selective dopamine transporter (DAT) inhibitor. Acutely, EC rats showed a greater, dose-dependent GBR 12935-induced increase in activity compared to IC rats; however, basal activity for EC rats was lower than for IC rats. After repeated GBR 12935, only EC rats exhibited behavioral sensitization. Kinetic analysis of DAT function in medial prefrontal cortex (mPFC) revealed that the maximal velocity of [3H]dopamine ([3H]DA) uptake in EC rats was less than in IC rats (4.9 +/- 0.6 and 7.7 +/- 0.6 pmol/min/mg, respectively), but not in striatum or nucleus accumbens. Furthermore, GBR 12935-induced inhibition of DAT function, [3H]GBR 12935 binding density and DA content in mPFC, striatum and nucleus accumbens were not different between EC and IC rats. However, dihydroxyphenylacetic acid content in mPFC was lower in EC than IC rats, whereas no differences were found in striatum and nucleus accumbens. These results suggest that EC-induced changes in activity may be due to decreased DAT function and decreased DA metabolism in the mPFC.

Lobeline inhibits nicotine-evoked [3H]dopamine overflow from rat striatal slices and nicotine-evoked 86Rb+ efflux from thalamic synaptosomes

The present study evaluated the interaction of lobeline with neuronal nicotinic acetylcholine receptors using two in vitro assays, [(3)H] overflow from [(3)H]dopamine ([(3)H]DA)-preloaded rat striatal slices and (86)Rb(+) efflux from rat thalamic synaptosomes. To assess agonist interactions, the effect of lobeline was determined and compared to S(-)-nicotine. To assess antagonist interactions, the ability of lobeline to inhibit the effect of S(-)-nicotine was determined. Both S(-)-nicotine (0.1-1 microM) and lobeline (>1.0 microM) evoked [(3)H] overflow from superfused [(3)H]DA-preloaded striatal slices. However, lobeline-evoked [(3)H] overflow is mecamylamine-insensitive, indicating that this response is not mediated by nicotinic receptors. Moreover, at concentrations (<1.0 microM) which did not evoke [(3)H] overflow, lobeline inhibited S(-)-nicotine (0.1-10 microM)-evoked [(3)H] overflow, shifting the S(-)-nicotine concentration-response curve to the right. S(-)-Nicotine (30 nM-300 microM) increased (EC(50) value=0.2 microM) (86)Rb(+) efflux from thalamic synaptosomes. In contrast, lobeline (1 nM-10 microM) did not evoke (86)Rb(+) efflux, and the lack of intrinsic activity indicates that lobeline is not an agonist at this nicotinic receptor subtype. Lobeline completely inhibited (IC(50) value=0.7 microM) (86)Rb(+) efflux evoked by 1 microM S(-)-nicotine, a concentration which maximally stimulated (86)Rb(+) efflux. Thus, the results of these in vitro experiments demonstrate that lobeline inhibits the effects of S(-)-nicotine, and suggest that lobeline acts as a nicotinic receptor antagonist.